DESCRIPTION: (Applicant's Description) Recent studies on the tumor suppressor genes BRCA1, BRCA2, ATM, and NBS1 indicate that they affect DNA double-strand break (DSB) repair by homologous recombination. There is a paucity of information about this repair pathway in humans, and the manner in which the repair process is modulated by the tumor suppressor proteins remains largely unknown. In order to understand how DSB repair helps maintain genomic stability and prevent cancer formation, it is necessary to first delineate the mechanism of the repair reaction. To achieve this goal, we will carry out a variety of mechanistic studies to dissect the DSB repair machinery in human cells. Existing evidence suggests that DSBs induced by ionizing radiation and chemicals are subjected to exonucleolytic processing, resulting in the formation of a single-stranded DNA region. Nucleation of recombination factors onto this single-stranded region leads to the formation of a recombinogenic complex, which then conducts a search for the intact chromosomal homolog. Invasion of the homolog by the initiating single- stranded DNA results in the formation of heteroduplex DNA. The human Mre11/Rad50/NBS1 nuclease complex which mediates DSB processing and various human RAD52 group proteins involved in heteroduplex DNA formation have been purified to near homogeneity. Functional studies will be carried out with the purified protein factors to elucidate the mechanisms of DNA break processing and heteroduplex formation, and to examine the coupling between the protein machines for break processing and heteroduplex formation. Extensive interactions and collaboration with investigators in the other Research Projects will focus on (i) the manner in which heteroduplex DNA formation is coupled to the repair DNA synthesis step (with Project 2), (ii) the regulation of repair complexes and machinery via ATM-dependent phosphorylation of repair proteins (with Project 3), and (iii) the function of BRCA1, BRCA2, MLB1, and RLB1 proteins in the assembly of repair protein complexes and machinery (with Project 4).